The genus Mycobacterium is responsible for more disease world-wide than all other bacterial genera combined. Mycobacteria are small, 0.5×1 μm, weakly gram-positive bacilli that have two characteristic bacteriologic features. First, the cell walls of these organisms contain very long chain fatty acid esters known as mycolic acids (lipids typically having chain lengths of 70 to 90 carbons). This unusual cell envelope allows the mycobacteria to be impervious to many antiseptic solutions and antibiotics. Secondly, the pathogenic mycobacteria have an unusually slow growth rate. Unlike typical bacterial pathogens such as E. coli or Staphylococcus aureus, which double in 20-30 minutes and grow to high densities overnight in culture media, M. avium and M. tuberculosis double approximately every 24 hours. Thus it can take three to four weeks to obtain a dense liquid culture or a visible colony of mycobacteria on a plate. The best known property of the genus is the ability of the bacteria to resist decolorization by weak acids after staining, hence the term “acid-fast bacilli” (Wayne “Cultivation of Mycobacterium tuberculosis for research purposes”, in Tuberculosis: Pathogenesis, Protection, and Control, ASM Press, Bloom BR (ed), Washington, D.C., 1994, pp. 73-84).
Within the genus Mycobacteria, there are a number of closely related species that have been grouped into complexes. Currently, over 60 species of mycobacteria have been well defined. Species other than M. tuberculosis and M. leprae have been termed the “environmental mycobacteria,” many of which are known pathogens. For example, M. kansasii is an important pulmonary pathogen, along with the M. avium complex.
The Mycobacterium avium complex (MAC) includes two species, M. avium and M. intracellulare. Though M. avium was originally identified as a pathogen of birds, it is now known that both M. avium and M. intracellulare are environmental saprophytes that survive well in soil, water, and food, and can be carried by animals (Inderlied et al., Clin Microbiol Rev 6:266-310, 1993; Shinnick and Good, Eur. J. Clin. Microbiol. Infect. Dis. 13:884-901, 1994).
MAC is an opportunistic, rather than innate, pathogen in humans; it causes disease primarily in immuno-compromised patients. MAC causes three classical disease syndromes: disseminated MAC, pulmonary MAC, and MAC cervical lymphadenitis, the most common of which is disseminated MAC (Horsburgh, “Epidemiology of Mycobacterium avium complex,” in Mycobacterium avium Complex Infection: Progress in Research and Treatment, Marcel Dekker, Inc., Korvick and Benson (eds), New York, 1996, pp. 1-22.
The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. Each of these species, except M. microti (a cause of rodent tuberculosis), causes tuberculosis in humans. Significantly, there are no environmental reservoirs of M. tuberculosis complex organisms, and only a few animals (cattle and occasionally deer) transmit tuberculosis to humans. Of all the culturable mycobacteria, only M. tuberculosis is an obligate pathogen.
M. tuberculosis causes a variety of disease syndromes depending upon the site of infection. The lung is the site of initial tuberculous infection and pulmonary tuberculosis remains the most common presentation, accounting for 85% of all tuberculosis in non-HIV infected patients. Pulmonary tuberculosis (TB) is the leading cause of death due to a single infectious organism in the world. There are 8 to 10 million new active cases of TB each year and approximately two million deaths. It is believed that one-third of the world's population, or 1.7 billion individuals, harbor latent tuberculosis (Centers for Disease Control and Prevention: Tuberculosis morbidity—United States, 1997. MMWR 47(13):253-7, 1998; Small et al., N. Engl. J. Med. 324:289-94, 1991).
Leprosy is caused by M. leprae, a slow growing obligate intracellular parasite. The disease is believed to occur as a result of a relatively specific defect in a patient's cell-mediated immune response to M. leprae. There are 11 to 12 million cases of leprosy worldwide; the United States has more than 5,500. Diagnosis of leprosy is based on physical findings plus skin scrapings and biopsy. Skin scrapings are evaluated by smearing the material onto a microscopic slide and histological staining (see below). Once diagnosed, leprosy can be treated using a combination anti-leprosy drug therapy (Jacobson, “Leprosy,” In: Textbook of Internal Medicine, Kelley et al. (eds.), J.B. Lippincott Co., 1989, pp. 1582-4).
In general, a combination therapy is essential for the effective treatment of TB or MAC disease, although different agents are used to treat the different infections. The identification of the species of mycobacteria involved in an infection is essential to determining the treatment protocol. For example, MAC organisms are resistant to many of the standard anti-mycobacterial drugs used for tuberculosis, including isoniazid, pyrazinamide, and often streptomycin.
The two laboratory techniques that have classically been used to detect a mycobacterial infection are the acid fast smear and cultivation of the organism. In addition, techniques have been developed to determine the species of the organism (e.g. DNA blotting techniques). Generally, these techniques are performed on a body fluid, such as sputum and blood (Heifets and Good, “Current laboratory methods for the diagnosis of tuberculosis,” in: Tuberculosis: Pathogenesis, Protection and Control. ASM Press, Bloom BR (ed), Washington, D.C., 1994, pp. 85-110).
Smear examinations (such as sputum smears) are usually performed within 24 hours after the specimen is submitted. Smear exams are more rapid but less sensitive than culture. Smears detect 10,000-100,000 organisms per ml of fluid or tissue. Although a negative smear does not rule out disease, it does suggest that the infection is light if it is present at all. Positive smears cannot differentiate between MAC, M. tuberculosis, or a host of other environmental acid-fast bacilli, and thus, they must be evaluated on the basis of the case history, as well as physical and laboratory findings.
The cultivation of mycobacteria is time-consuming. It usually takes three to four weeks to obtain colonies on standard semi-solid medium. Most laboratories in the U.S. use the BACTEC™ radiometric mycobacterial growth system, which detects the growth of mycobacteria by their metabolic conversion of radioactive palmitic acid in the liquid growth medium into radioactive gaseous CO2. The BACTEC™ system frequently yields positive cultures with 8-15 days. However, the BACTEC™ system does not differentiate between M. tuberculosis infection, MAC, and other slow-growing mycobacterial infections (Kent and Kubica, Public Health Mycobacteriology: a Guide for the Level III Laboratory, US Dept. of Health and Human Services, Atlanta, Ga., 1985; Siddiqi, Radiometric (BACTEC) tests for slowly growing mycobacteria, in: Clinical Microbiology Procedures Handbook, Vol. 1, Isenberg HD (ed), American Society for Microbiology, Washington, D.C., 1992). This is a serious clinical problem because appropriate therapy (and the need for precautionary respiratory isolation) often depends on the species of Mycobacterium that is present.
Determining the Mycobacterium species present in a sample has been accomplished in the past by removing a small portion of the organism that has grown in the BACTEC™ bottle, and performing a series of DNA hybridization tests with species-specific probes. The DNA probe test (e.g. AccuProbe Kit) takes approximately four hours and can usually be performed a few days after detection of the organism in the BACTEC™ system (Centers for Disease Control and Prevention: Nucleic Acid Amplification Tests for Tuberculosis. MMWR 45:950-952, 1996). Alternatively, a battery of time consuming biochemical and selective medium growth tests can be performed in order to speciate acid fast isolates. However, these procedures often require growth of the bacteria, a process which is time consuming because of the slow growth of mycobacterial organisms.
Nucleic acid amplification methods have been used to identify M. tuberculosis in a sample (e.g. U.S. Pat. No. 5,811,269; U.S. Pat. No. 5,736,365). However, there remains a need in the art for a rapid method for detecting the other species of Mycobacterium in samples, so that appropriate therapy and any necessary precautions can be initiated and/or terminated as soon as possible. In addition, a need exists for a method to provide rapid identification of not just one species, but multiple species of Mycobacterium, particularly those species that are pathogenic, such as known human pathogens.